Short hairpin RNA targeting Notch2 inhibits U87 human glioma cell proliferation by inducing cell cycle arrest and apoptosis in vitro and in vivo

Li,Xuezhen; He,Xin; Tian,Wei; Wang,Jianzhen

doi:10.3892/mmr.2014.2661

Short hairpin RNA targeting Notch2 inhibits U87 human glioma cell proliferation by inducing cell cycle arrest and apoptosis in vitro and in vivo

Authors:
- Xuezhen Li
- Xin He
- Wei Tian
- Jianzhen Wang
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Affiliations: Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, P.R. China, Department of Neurosurgery, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing 100093, P.R. China, Department of Neurosurgery, Handan Central Hospital, Handan 056001, P.R. China, Department of Neurosurgery, General Hospital of Chinese People's Armed Police Forces, Beijing 100039, P.R. China
Published online on: October 15, 2014 https://doi.org/10.3892/mmr.2014.2661
Pages: 2843-2850
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

Notch signaling has been reported to be oncogenic or tumor suppressive, depending on the tissue context. To investigate the effects of Notch2 knockdown on U87 human glioma cell proliferation in vitro and in vivo, and the associated mechanisms, U87 cells were stably transfected with p green fluorescent protein (GFP)‑V‑RS Notch2 short hairpin (sh) RNA plasmid and pGFP‑V‑RS scramble‑shRNA plasmid. The former was referred to as the Notch2‑shRNA group and the latter as the negative‑shRNA group. mRNA and protein expression, cell proliferation, cell cycle and apoptosis were measured by reverse transcription‑polymerase chain reaction, western blot analysis, 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide analysis and flow cytometry using propidium iodide, respectively. Tumor volume, tumor weight and cumulative survival rate were determined in a nude mouse xenograft tumor model. Notch2 mRNA and protein expression in the Notch2‑shRNA group were reduced by 87.6 and 94.5% compared with the negative‑shRNA group (P<0.001). Notch2 knockdown significantly inhibited U87 cell proliferation after three days of culture (P<0.05). Notch2 silencing induced cell cycle arrest at G0/G1 phase by upregulation of p21 protein expression and downregulation of mini chromosome maintenance complex 2 and cyclin‑D1 protein expression. Furthermore, knockdown of Notch2 also induced U87 cell apoptosis. On day 50 after inoculation, tumor weight in the Notch2‑shRNA group was significantly lower than that in the negative‑shRNA group (0.55±0.10 vs. 1.23±0.52 g; P<0.01). The cumulative survival rate was significantly longer in the Notch2‑shRNA group compared with the negative‑shRNA group (log rank test P=0.01). In conclusion, Notch2 silencing inhibited U87 glioma cell proliferation by inducing cell cycle arrest and apoptosis in vitro and in vivo. Thus, Notch2 may be a key therapeutic target for the treatment of glioma.

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